Method for sequencing nucleic acid molecules
First Claim
1. A method of sequencing a target nucleic acid molecule comprising:
- (a) providing a mixture comprising the target nucleic acid molecule, a primer complementary to the target nucleic acid molecule, a nucleic acid polymerizing enzyme, and a plurality of types of nucleotides or nucleotide analogs to be incorporated into a growing nucleic acid strand at an active site complementary to the target nucleic acid, wherein the target nucleic acid molecule and/or the nucleic acid polymerization enzyme is attached to a support;
(b) subjecting the mixture of step (a) to a polymerization reaction under conditions suitable for formation of the growing nucleic acid strand by template-directed extension of primer; and
(c) optically identifying a time sequence of incorporation of nucleotides or nucleotide analogs into the growing nucleic acid strand at the active site while the template-directed extension of primer is taking place, and thereby determining the sequence of said target nucleic acid.
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Abstract
The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
457 Citations
39 Claims
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1. A method of sequencing a target nucleic acid molecule comprising:
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(a) providing a mixture comprising the target nucleic acid molecule, a primer complementary to the target nucleic acid molecule, a nucleic acid polymerizing enzyme, and a plurality of types of nucleotides or nucleotide analogs to be incorporated into a growing nucleic acid strand at an active site complementary to the target nucleic acid, wherein the target nucleic acid molecule and/or the nucleic acid polymerization enzyme is attached to a support; (b) subjecting the mixture of step (a) to a polymerization reaction under conditions suitable for formation of the growing nucleic acid strand by template-directed extension of primer; and (c) optically identifying a time sequence of incorporation of nucleotides or nucleotide analogs into the growing nucleic acid strand at the active site while the template-directed extension of primer is taking place, and thereby determining the sequence of said target nucleic acid. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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2. A method of sequencing a target nucleic acid molecule comprising:
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subjecting the target nucleic acid molecule to a polymerization reaction to yield a growing nucleic acid strand that is complementary to the target nucleic acid molecule in the presence of a reaction mixture of plurality of types of nucleotides or nucleotide analogs, wherein the target nucleic acid molecule and/or the nucleic acid polymerization enzyme is attached to a support; and optically identifying during said subjecting step, the nucleotide or nucleotide analog incorporated into the growing strand to determine a time sequence of incorporation of said plurality of types of nucleotides or nucleotide analogs in said reaction mixture into the growing nucleotide strand.
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Specification